Abstract

The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding–associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.

Introduction

Complement is a proteolytic cascade that can be activated via three routes: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). Ca2+-dependent complexes consisting of pattern recognition molecules (PRMs) and associated serine proteases initiate the CP and LP. The most abundant protease of the LP is mannose-binding lectin–associated serine protease (MASP)-1. When the LP is triggered, MASP-1 autoactivates first and then it cleaves MASP-2 in normal human serum (NHS) (1). MASP-2 cleaves C4, whereas C2 is cleaved by MASP-1 and MASP-2. The LP generates the same type of C3 convertase (C4b2a) as the CP. The mechanism of AP activation is quite different, because there is no known PRM of the AP. Properdin, the positive regulator of the AP, was suggested to act as a PRM, but this issue is controversial (2). The AP is initiated when C3b or C3b-like molecules emerge near a surface. C3b can covalently bind to the surface and it binds complement factor B (FB). C3b-bound FB is cleaved by complement factor D (FD), a serine protease that circulates predominantly in the active form. The resulting C3bBb is the AP C3 convertase, which converts more C3 into C3b, providing a positive feedback. Properdin, the only known positive regulator of complement, stabilizes the AP C3 convertase (3). The AP can be initiated independently of the CP or the LP. According to the prevailing “tickover” hypothesis, the reactive thioester bond of C3 slowly hydrolyzes in the fluid phase, and the emerging C3b-like molecule, C3(H2O), binds FB. After having been cleaved by FD, the resulting C3(H2O)Bb complex acts as a C3 convertase. If this fluid-phase convertase is generated near a surface, the nascent C3b molecules can bind there covalently and initiate the amplification process. Endogenous cells are protected from excessive complement activation by cell surface–anchored and fluid-phase inhibitors. The most important AP inhibitor is complement factor H (FH), which can bind to self-surface–deposited C3b and destroy it by recruiting the serine protease, factor I.

The three activation pathways had been considered independent activation routes; however, evidence has begun to accumulate suggesting various cross-talks. Recently, an important connection between the LP and the AP has been demonstrated. Activated MASP-3 is a protease responsible for activating pro-FD in the circulation (4–6); in fact, it is the only protease doing so in resting human blood (7).

In this article, we describe an unexpected link between the LP and the AP, demonstrating a novel function of MASP-1. On certain activator surfaces, MASP-1 has been found to be indispensable for AP activation in the absence of Ca2+. This phenomenon might be of great importance for protection against invading microorganisms, but it also might contribute to uncontrolled complement activation, leading to severe symptoms.

Materials and Methods

Reagents

All commonly used reagents were purchased from Sigma-Aldrich or Merck, unless otherwise indicated. Human FB was purchased from Merck (Calbiochem brand). Human C1 inhibitor (Berinert P) was from CSL Behring. LPS from three Gram-negative bacterial strains (Salmonella typhimurium [L6511], Escherichia coli [L8274], and Pseudomonas aeruginosa [L9143]) was purchased from Sigma-Aldrich. Zymosan A from Saccharomyces cerevisiae was from Santa Cruz Biotechnology (catalog number sc-258367A).

Human serum, anti–MASP-1 Ab, and purified proteins

NHS collected from 10 healthy volunteers was pooled and stored at −80°C. Production of Ab against the serine protease domain (SP) of MASP-1 (αM1-SP) was described previously (6). MASP-1–depleted serum was prepared by running NHS through an anti–MASP-1 column twice. The catalytic fragment of human MASP-1 (rMASP-1cf) was purified as described (8). The N-terminal fragment of MASP-1 (M1_D1-3) was purified as described (9). The MASP-1–specific inhibitor (SGMI-1) and the MASP-2–specific inhibitor (SGMI-2) were produced as described (10). The MASP-3–specific inhibitor (TFMI-3) was prepared as described (7). Recombinant FD was produced as described (5). The recombinant serpin domain of human C1 inhibitor was produced as described (11). C3 was purified from human EDTA plasma (12). C3b was generated from C3 using trypsin (T8003; Sigma-Aldrich), which was later removed chromatographically from C3b.

Inhibition assays

The activity of the AP was measured in the presence of MASP-1–specific inhibitors (SGMI-1, 1.25–20 μM; αM1-SP, 2.1–133 nM; M1_D1-3, 0.06–7.3 μM), specific inhibitors of other complement proteases (SGMI-2, 1.25–20 μM; TFMI-3, 0.29–30 μM), full-length C1 inhibitor (0.35–11.3 μM), and the serpin domain of C1 inhibitor (1.14–9.1 μM). The effect of the above-mentioned molecules was followed through the decrease in C3 deposition.

Inhibition of ongoing C3 deposition

NHS (diluted 6-fold) was incubated in separate wells of LPS-coated microtiter plates for 45 min at 37°C. At different time points during this period, the same volume (4.8 μl) of SGMI-1 or FUT-175 inhibitor was added to the individual reactions to reach a final concentration of 20 or 100 μM, respectively. The same volume of serum dilution buffer was also added to some wells as negative control. After a total of 45 min, serum was discarded, the microtiter plate was washed, and the deposited C3 fragments were measured similarly as described above.

Statistical analysis

Statistical analysis was performed with Origin 6.0 (OriginLab, Northampton, MA) software. The Student t test was used to determine significant differences between the control and treated samples. Two levels of significance were used: *p < 0.05 and **p < 5 × 10−9.

Results and Discussion

To reveal the individual roles of MASPs in complement activation, we previously developed selective and potent inhibitors against MASP-1 (SGMI-1), MASP-2 (SGMI-2), and MASP-3 (TFMI-3) (7, 10). In this study, we used these inhibitors to monitor activation of the AP using NHS (diluted 6-fold) in Mg2+-EGTA buffer. We set up ELISA assays for measuring C3 deposition. Two activators are used routinely to measure AP activation on plates: bacterial LPS and yeast zymosan. To our great surprise, the MASP-1–specific inhibitor (SGMI-1), at a concentration of 10 μM, efficiently inhibited C3 deposition, and it caused near-complete abrogation, at 20 μM, on LPS-coated surfaces (IC50 = 6.2 μM) (Fig. 1A). Contrary to that, only a partial (∼20%) inhibitory effect was detected on zymosan-coated surfaces, even at the highest inhibitor concentration (20 μM) (Fig. 1A). SGMI-2, the MASP-2–specific inhibitor, caused only marginal inhibition of C3 deposition on the LPS-coated surface and had no effect on the zymosan-coated one (Fig. 1B). Inhibition of MASP-3 using TFMI-3 did not influence LPS- or zymosan-induced AP activation (Fig. 1C). These results imply that MASP-1 has a key role in AP activation on LPS-coated surfaces. The IC50 value for the inhibition of LPS-induced AP activation by SGMI-1 (6.2 μM) is much higher than the value (20 nM) calculated by considering only the 7 nM Kd value of the enzyme/inhibitor complex and the molar concentration of MASP-1 (25 nM) in the assay. We suggest that the complexity of the serum and the multistep nature of the ELISA assay that we used result in an IC50 value significantly higher than expected.

Effect of inhibition of MASPs on LPS- and zymosan-induced AP complement activation. AP activation on different activation surfaces was followed by the detection of C3 deposition. The experiments were performed using NHS diluted 6-fold in Mg2+-EGTA buffer. The plates were coated with bacterial LPS (S. typhimurium) or zymosan (S. cerevisiae) and incubated with the serum containing different inhibitors. The MASP-1–specific inhibitor (SGMI-1) completely inhibited AP activation on the LPS-coated surface (A), whereas the MASP-2–specific (B) and the MASP-3–specific (C) inhibitors showed little or no effect. C3b deposition reflects deposition of various C3 fragments. Results are representative of at least three independent experiments.

MASP-1 inhibitors having different mechanisms of action elicit the same effect on AP activation

Because the inhibitory effect of SGMI-1 on AP activation was detected only at a relatively high inhibitor concentration, we had to rule out the possibility that the observed effect of SGMI-1 is exerted through its weak nonspecific inhibition of a protease or proteases other than MASP-1. To this end, we applied MASP-1 inhibitors having different mechanisms of action. αM1-SP showed a concentration-dependent inhibitory effect on C3 deposition on the LPS-coated surface, whereas it had only a marginal effect on the zymosan-coated surface (Fig. 2A). At 130 nM, it completely prevented LPS-induced AP activation. This Ab is also an efficient LP inhibitor (Supplemental Fig. 1).

M1_D1-3 is responsible for dimerization and for binding to the PRMs; therefore, we investigated its effect on AP activity. It has already been shown that M1_D1-3 is able to attenuate LP activation in NHS through displacing the MASPs from the PRMs (9). M1_D1-3 behaved similarly to SGMI-1 in the AP assay system; it inhibited AP activation on the LPS-coated surface in a concentration-dependent manner but showed only limited effect on the zymosan-coated plate (Fig. 2B). This result suggests that binding of MASP-1 via its N-terminal region to a PRM or to other unknown molecules plays an important role in AP activation triggered by LPS. The binding molecule cannot be mannose-binding lectin (MBL), because MBL binding to carbohydrates is strictly Ca2+ dependent. It is very likely that PRM–MASP complexes are largely intact at physiological salt concentration in the presence of Mg2+-EGTA (13, 14). We tried to demonstrate MASP-1 binding to LPS-coated wells using the MASP-1–specific Ab, but we could not detect any signal (data not shown). This could mean that the binding is weak or transient, but it is still enough to support AP activation on the LPS surface. It should be noted that M1_D1-3 is not strictly MASP-1 specific; it displaces all MASPs and MBL-associated proteins from the PRMs.

The natural inhibitors of MASPs are serpins. C1 inhibitor is an efficient inhibitor of the LP in NHS (15). We tried to prevent MASP-1–mediated AP activation using C1 inhibitor. We did not get any inhibition of C3 deposition on LPS-coated plates using natural C1 inhibitor isolated from human blood (Fig. 2C). Contrary to that, we detected concentration-dependent inhibition using the recombinant serpin domain of C1 inhibitor (Fig. 2C). C1 inhibitor is an atypical serpin, because, in addition to the serpin domain, it possesses a heavily glycosylated N-terminal domain. Our results indicate that, in the LPS-induced AP-activation system the N-terminal domain somehow prevents interaction between MASP-1 and C1 inhibitor. In contrast, the single globular serpin domain of C1 inhibitor has free access to active MASP-1 under these conditions. Similar observations were reported earlier using StcE-treated C1 inhibitor. StcE is a metalloprotease secreted by E. coli O157:H7 that can remove the N-terminal domain of the C1 inhibitor. The resulting truncated C1 inhibitor efficiently inhibits endothelial cell–bound kallikrein, whereas the full-length C1 inhibitor fails to do so (16). This suggests that the N-terminal domain of C1 inhibitor might have an important regulatory function; it hinders the rapid and efficient interaction between the serpin and the target protease on a surface of a pathogen promoting complement activation, whereas the full-length molecule can successfully prevent spontaneous activation of complement proteases in the fluid phase. Neither the full-length C1 inhibitor nor the serpin domain could attenuate AP activation on the zymosan-coated surface. Fig. 2D summarizes the results of three independent measurements with each inhibitor on LPS- or zymosan-coated surfaces.

To corroborate the results obtained using various inhibitors in NHS, we checked the ability of MASP-1–depleted serum to activate the AP in our assay system. In accordance with the above results, MASP-1–depleted serum showed very low AP activity on the LPS-coated surface, whereas the AP activity was only partially compromised on the zymosan-coated surface (Fig. 3A). Taken together, these results show that MASP-1 has a central role in LPS-induced AP activation.

C3 deposition in the presence of MASP-1–depleted serum and C3 deposition on different surfaces. (A) MASP-1 depletion severely reduced C3 deposition in the case of LPS-induced AP activation, whereas it had only a limited effect on zymosan-induced AP activation. MASP-1–depleted serum was applied on LPS- or zymosan-coated surfaces. Results are representative of at least three independent experiments. (B) Inhibition of MASP-1 proteolytic activity blocks AP activation on different surfaces. The inhibitory effect of SGMI-1 (20 μM) was tested on AP activation triggered by various activation surfaces. Microtiter wells were coated with bacterial LPS isolated from different strains (S. typhimurium, E. coli, or P. aeruginosa). SGMI-1 effectively blocked AP activation in all cases. The AP activation induced by mannan was also inhibited by SGMI-1. C3b deposition reflects deposition of various C3 fragments. Results are representative of at least three independent experiments.

Surface dependence of MASP-1–mediated AP activation

To refine our knowledge about the surface requirement of MASP-1–mediated AP activation, we tested various AP activators. In addition to the LPS type (S. typhimurium) that we used in the experiments above, we checked LPS preparations from other bacteria. The E. coli and P. aeruginosa LPS preparations gave the same results: high-level C3 deposition that could be almost completely inhibited by 20 μM SGMI-1 (Fig. 3B). Selander et al. (17) demonstrated that a mannan-coated surface induces efficient AP activation in Mg2+-EGTA buffer. Using the same activation surface, we showed that mannan-induced AP activation can also be blocked by our MASP-1–specific inhibitor (Fig. 3B). Finally, we tested the lysis of rabbit erythrocytes, a frequently used method to measure AP activity. In this assay system, human serum (diluted 18-fold) readily lysed rabbit erythrocytes, and the lysis could not be attenuated by SGMI-1, even at high concentration (20 μM; data not shown). Our results are in line with those of Degn et al. (18), who used MASP-1/3–deficient serum from a patient with 3MC syndrome and measured efficient lysis of rabbit erythrocytes.

Inhibition of MASP-1 attenuates C3 deposition in progress

Theoretically, AP activation can be divided into distinct parts: the first part is the initiation lag phase, when the first few C3b molecules deposit on the activation surface, and the second part is the amplification phase, when the surface-deposited C3b molecules serve as a base for generating additional C3 convertase complexes building up the positive-feedback loop. We also studied to which phase of the AP activation MASP-1 contributes. To this end, we launched parallel AP-activation experiments; NHS was incubated on an LPS-coated surface for different times to let the C3b deposition reach a certain level and then we added the same volume of SGMI-1, FUT-175, or buffer to the individual reactions. FUT-175 is a broad-spectrum serine protease inhibitor that inhibits all complement proteases. In concordance with previous results (19), the time course of C3 deposition shows an initial lag phase (5–6 min), followed by a rapid amplification phase (Fig. 4). SGMI-1 reduced C3 deposition at all phases of AP activation. Even in the presence of surface-deposited C3b, SGMI-1 attenuated additional deposition of C3b, although not as efficiently as the broad-spectrum serine protease inhibitor FUT-175. These results suggest that MASP-1 is necessary for maintaining efficient amplification of LPS-induced AP activation; however, it does not necessarily preclude that it is also important for the initiation.

Inhibition of MASP-1 attenuates the amplification phase of LPS-induced AP activation. NHS (diluted 6-fold in Mg2+-EGTA buffer) was transferred onto LPS-coated wells and incubated at 37°C. At different time points, MASP-1–specific inhibitor (SGMI-1; 20 μM), broad-specificity serine protease inhibitor (FUT-175; 100 μM), or buffer was added, and C3 deposition was measured. SGMI-1 and FUT-175 prevented further C3 deposition, even when the surface was partially covered with C3b. C3b deposition reflects deposition of various C3 fragments. Results are representative of at least three independent experiments.

MASP-1 does not cleave FB

We have assessed the direct proteolytic action of MASP-1 on components of the AP. Previously, we studied the ability of MASP-1 to cleave C3 and pro-FD (5, 20). In this study, we assessed whether MASP-1 can cleave FB. We mixed FB with C3b and rMASP-1cf, incubated the mixture at 37°C, and followed the digestion using SDS-PAGE (Fig. 5). We found that 50 nM MASP-1 was not able to cleave C3b-bound FB. Although some Bb fragment appeared after 1.5 h, the band was not significantly stronger than for the negative control. Under similar conditions, 0.25 nM FD cleaved FB with high efficiency. We also demonstrated that SGMI-1 does not inhibit FD-mediated FB cleavage significantly (Fig. 5). Taken together, it seems unlikely that MASP-1 exerts its proteolytic effect directly on the known components of the AP. Instead, it may cleave a still unidentified factor and, thereby, promote AP activation.

Effect of MASP-1 and SGMI-1 on the cleavage of C3b-bound FB. C3b (1.23 μM) was mixed with FB (1.07 μM), and the resulting proconvertase complex was incubated with rMASP-1cf (50 nM) or with FD (0.25 nM) in the presence or absence of SGMI-1 (20.5 μM). After incubation at 37°C for 1.5 h, the reactions were stopped, and cleavage of FB was analyzed on reducing 10% SDS-PAGE. rMASP-1cf was unable to cleave FB, whereas, under the same conditions, FD was very efficient. FD-mediated FB cleavage could not be significantly prevented by SGMI-1.

Concluding remarks

Our observations are in accordance with previous results from the literature. The finding that the mechanism of AP activation depends on the nature of the activator was reported by Kimura et al. (21). They found that properdin was indispensable for LPS-induced AP activation in mouse serum, whereas properdin deficiency impaired zymosan-induced AP activation only minimally. The reason for the differential requirement of properdin by different AP activators is unknown, but it is intriguing that we got the same surface dependence for MASP-1–supported AP activation using NHS. It is plausible to assume that the extent of AP activation on certain surfaces depends on the balance between properdin- and MASP-1–mediated promotion and FH-dependent inhibition. It is very likely that FH binds to LPS more strongly than to zymosan. It is possible that there is a connection between the requirement of properdin and MASP-1 for the activity of the AP on certain activation surfaces, but further experiments are needed to investigate this possibility.

Although anti-LPS Abs may play a role in AP activation, removal of Abs from the serum did not influence MASP-1 dependence on LPS-induced AP activation (data not shown).

Selander et al. (17) studied the contribution of MBL to AP activation. Although they concluded that MBL alone (without the contribution of MASPs) triggers C3 deposition on solid-phase mannan or mannan-rich LPS in C2-deficient serum, their experimental data clearly indicated that MASP-1 has a role in C2-bypass activation. In their experiments, serum fractions containing MBL/MASP-1 complexes were able to activate C3 in the absence of C2, whereas other MBL/MASP complexes failed to do so. The extent of the C3 deposition depended solely on the MASP-1 content of the MBL/MASP complexes. We also observed vigorous C3 deposition on the mannan-coated surface in the presence of Mg2+-EGTA, which was entirely MASP-1 dependent. MBL cannot bind to mannan under these conditions; however, other PRMs may retain this ability in the absence of Ca2+ (22). It is possible that, in the presence of Ca2+, where MBL binds to its target, the AP-promoting activity of PRM–MASP-1 complexes is even higher and may contribute significantly to the defense against Gram-negative bacteria. Selander et al. (17) speculate that MASP-1 may act on C3 indirectly by recruiting other serum proteases that cleave C3. Our results are also reconcilable with this hypothesis especially, because we demonstrated earlier that MASP-1 is an atypical complement serine protease with relatively broad substrate specificity (23).

The fact that MASP-1 is necessary for LPS-induced AP activation, but not for zymosan-induced AP activation, suggests that the mechanism of AP activation differs in the case of the two different activators. Further studies are required to clarify the exact molecular mechanism of complement activation on these activator surfaces.

In conclusion, we discovered a novel role for MASP-1 in complement activation. Although it was suggested earlier by several investigators that MASP-1 might contribute to AP activation, no solid evidence has been presented. The activator-specific requirement of MASP-1 in AP activation indicates a tight interaction between the complement-activation routes, which were previously considered independent.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

This work was supported by National Research, Development and Innovation Office/Hungarian Scientific Research Fund Grants K108642, K119374, and K119386 and by the MedInProt Protein Science Research Synergy Program of the Hungarian Academy of Sciences.

. 2013. The control of the complement lectin pathway activation revisited: both C1-inhibitor and antithrombin are likely physiological inhibitors, while α2-macroglobulin is not.Mol. Immunol.54: 415–422.